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Research article2007Peer reviewed

Structural analysis of altered large-subunit loop-6/carboxy-terminus interactions that influence catalytic efficiency and CO2/O-2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Karkehabadi, Saeid; Satagopan, Sriram; Taylor, Thomas C.; Spreitzer, Robert J.; Andersson, Inger

Abstract

The loop between a-helix 6 and beta-strand 6 in the alpha/beta-barrel of ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in discriminating between CO2 and O-2. Genetic screening in Chlamydomonas reinhardtii previously identified a loop-6 V331A substitution that decreases carboxylation and CO2/O-2 specificity. Revertant selection identified T342I and G344S substitutions that restore photosynthetic growth by increasing carboxylation and specificity of the V331A enzyme. In numerous X-ray crystal structures, loop 6 is closed or open depending on the activation state of the enzyme and the presence or absence of ligands. The carboxy terminus folds over loop 6 in the closed state. To study the molecular basis for catalysis, directed mutagenesis and chloroplast transformation were used to create T342I and G344S substitutions alone. X-ray crystal structures were then solved for the V331A, V331A/ T342I, T342I, and V331A/G344S enzymes, as well as for a D473E enzyme created to assess the role of the carboxy terminus in loop-6 closure. V331A disturbs a hydrophobic pocket, abolishing several van der Waals interactions. These changes are complemented by T342I and G344S, both of which alone cause decreases in CO2/O-2 specificity. In the V331A/T342I revertant enzyme, Arg339 main-chain atoms are displaced. In V331A/G344S, a-helix 6 is shifted. D473E causes disorder of the carboxy terminus, but loop 6 remains closed. Interactions between a transition-state analogue and several residues are altered in the mutant enzymes. However, active-site Lys334 at the apex of loop 6 has a normal conformation. A variety of subtle interactions must be responsible for catalytic efficiency and CO2/O-2 specificity.

Published in

Biochemistry
2007, Volume: 46, number: 39, pages: 11080-11089

      SLU Authors

    • Karkehabadi, Saeid

      • Department of Molecular Biology, Swedish University of Agricultural Sciences

      • Taylor, Tom

        • Department of Molecular Biology, Swedish University of Agricultural Sciences

        • Andersson, Inger

          • Department of Molecular Biology, Swedish University of Agricultural Sciences

        UKÄ Subject classification

        Structural Biology
        Biochemistry and Molecular Biology

        Publication identifier

        DOI: https://doi.org/10.1021/bi701063f

        Permanent link to this page (URI)

        https://res.slu.se/id/publ/31353